LGI1 Autoantibodies Enhance Synaptic Transmission by Presynaptic Kv1 Loss and Increased Action Potential Broadening

Background and Objectives Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. Methods We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. Results LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. Discussion Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.


Introduction
Autoimmune encephalitis is a growing group of diseases caused by autoantibodies against various neuronal antigens, collectively leading to severe mental and behavioral disorders. 1,2Autoimmune encephalitis with a predominant phenotype of limbic system involvement (so-called limbic encephalitis) primarily affects the mesial temporal lobe, hippocampus, and amygdala and is characterized by focal and generalized seizures and limbic dysfunction including mood changes and amnesia.The most frequent type of limbic encephalitis is caused by autoantibodies against the neuronal protein leucine-rich glioma inactivated 1 (LGI1) 2 resulting in characteristic faciobrachial dystonic and generalized seizures together with amnestic deficits.[9] LGI1 has 2 main domains, a N-terminal leucine-rich repeat (LRR) and a C-terminal epitempin (EPTP) domain. 102][13] LGI1-ADAM22 heterodimers have been suggested to dimerize in the synaptic cleft through an LRR-EPTP interaction, thus linking LGI1-ADAM22s within presynaptic and postsynaptic membranes to form a transsynaptic-tetrameric complex. 12ADAM22 receptors have been reported to interact directly or indirectly with both, presynaptic proteins including CASK, SAP97, and various poreforming or accessory K v 1 and Ca v channel subunits, and the postsynaptic neurotransmitter receptor scaffold including PSD95 and glutamate receptors. 14,15In addition, LGI1 was found to be critical for potassium channel expression 16 and function. 17The transsynaptic LGI1-ADAM22 complex was therefore proposed as a key component controlling presynaptic transmitter release to postsynaptic receptors. 18 study the function of LGI1 at synapses and the consequences of disturbed LGI1 signaling, 2 main approaches have been adopted.First, genetically modified cell lines or animal models were used either that overexpressed LGI1, 19 did not express LGI1, 11,20,21 or that harbored genetic variations of LGI1 associated with inherited epilepsy. 11,19,22,23In most of these studies, LGI1 overexpression enhanced and LGI1 loss reduced the postsynaptic α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor response and receptor clustering. 11,13,22,24Furthermore, LGI1 loss increased the presynaptic neurotransmitter release. 16,19,21Second, synaptic LGI1 function was studied using patient-derived polyclonal LGI1 autoantibodies [25][26][27][28] or domain-specific monoclonal autoantibodies. 29,30Reminiscent of genetically induced LGI1 loss, treatment with autoantibodies reduced postsynaptic AMPA receptors in primary hippocampal cultures 26 and acute hippocampal brain slices. 27Furthermore, recent evidence indicates that LGI1 autoantibodies increase presynaptic release probability and overall synaptic strength [27][28][29] with strengthening paralleled by potassium channel loss. 27,30However, the mechanism by which LGI1 autoantibodies strengthen presynaptic neurotransmitter release and the responsible molecular domains remain elusive.
Here, we combined electrophysiologic somatic and subcellular presynaptic recordings from cultured hippocampal neurons with stimulated emission depletion (STED) microscopy, 31 expansion microscopy (ExM) together with structured illumination microscopy (SIM), 32,33 and electron microscopy to identify mechanisms involved in the LGI1 autoantibody-mediated increase in presynaptic release.We find that polyclonal LGI1 autoantibodies increase presynaptic release probability independent of calcium channels.By contrast, polyclonal LGI1 autoantibodies reduce presynaptic K v 1.1 and K v 1.2 channels and lead to increased action potential broadening, an effect replicated by monoclonal EPTP, but not LRR autoantibodies.

Standard Protocol Approvals, Registrations, and Patient Consents
Animal experiments were performed in accordance with the ARRIVE guidelines, and animals were handled according to Glossary AIS = axon initial segment; EPSCs = excitatory postsynaptic currents; LGI1 = leucine-rich glioma inactivated 1; LRR = leucinerich repeat; PPR = paired-pulse ratio; SIM = structured illumination microscopy; STED = stimulated emission depletion.

Data Availability
Data that support the findings of this study are available from the corresponding author on reasonable request.

Results
LGI1 Autoantibodies Induce a Dose-Dependent Increase in Synaptic Release Probability We first investigated the effect of polyclonal LGI1 autoantibodies on excitatory transmission in primary dissociated hippocampal cultures.Polyclonal LGI1 autoantibodies were obtained from the serum of 3 patients with LGI1 encephalitis and high titer of LGI1 antibodies and used as a pooled IgG fraction. 27Cultures were incubated with patient-derived polyclonal serum LGI1 autoantibodies included in the growth medium for 7 days (LGI1-7d; with a second dose applied 1 day before recordings), with LGI1 autoantibodies for 1 day only (LGI1-1d), or with patient control antibodies without antineuronal reactivity.We then recorded pharmacologically isolated excitatory postsynaptic currents (EPSCs) in somatic whole-cell voltage-clamp recordings evoked by external stimulation.As a measure of presynaptic release probability, we determined the paired-pulse ratio (PPR), which is largely independent of postsynaptic strength, synapse number, and neuronal morphology. 34EPSCs in control autoantibody-incubated neurons showed facilitation at frequencies of 20 Hz, reflected in PPR >1.Treatment with LGI1 autoantibodies reduced PPRs in a dose-dependent manner, indicating that LGI1 autoantibodies increase the synaptic release probability (Figure 1 Similarly, LGI1 autoantibodies reduced PPRs at 50 Hz stimulation (eFigure 1).To study autoantibody-induced changes in synaptic transmission in more detail, we analyzed short-term plasticity during evoked EPSC trains (50 EPSCs at 20 Hz; Figure 1C).LGI1 autoantibody treatment suppressed facilitation and induced faster and stronger depression of excitatory currents, in line with higher presynaptic release probability on autoantibody treatment (Figure 1D  However, this is not surprising because the total EPSC amplitude besides release probability also depends on, e.g., postsynaptic strength, synapse number, and neuronal morphology, which may change on LGI1 loss.Because 7-day antibody treatment more robustly affected synaptic transmission than 1-day treatment, we adopted the 7-day treatment for subsequent analyses. 5][36] Furthermore, proteome studies indicate interactions between the LGI1-receptor ADAM22 and calcium channels. 18,37Therefore, a straightforward explanation for the autoantibody-induced increase in release probability could be an increased presynaptic calcium influx due to either higher calcium channel abundance or faster channel gating.To first test whether LGI1 autoantibodies affected presynaptic calcium channel abundance, we performed STED imaging of Ca v 2.1 channels, which is one of the main calcium channel types at hippocampal synapses. 38,39Ca v 2.1 fluorescence signal intensities were quantified at excitatory presynapses (labeled by the vesicular glutamate transporter vGlut1, eFigure 2) and excitatory active zones (labeled by Bassoon within vGlut1-positive presynapses, Figure 2A).
To analyze the effect of LGI1 autoantibodies on Ca v 2.1 channels within presynaptic active zones in more detail, we performed freeze-fracture replica immunoelectron microscopy of hippocampal presynapses from mice chronically infused with patient-derived polyclonal serum LGI1 autoantibodies using intraventricular osmotic pumps.LGI1 antibodies infused by osmotic pumps penetrated into the tissue and particularly into the hippocampus where they bound to their target antigen in contrast to a control antibody (eFigure 3).Antibody-treated mice did not develop obvious epileptic symptoms.We first analyzed dentate gyrus perforant path-granule cell synapses (Figure 2C) because LGI1 expression is highest in the dentate gyrus 17 and transmission is affected presynaptically on genetic alteration of LGI1 19 or LGI1 autoantibodies. 27Chronic LGI1 autoantibody infusion did not affect active zone Ca v 2.1 channel density (Figure 2D; median [IQR] Ca v 2.1 particle density per μm 2 : 248 [186-444] and 220 [156-386], 96 and 117 active zones for control and LGI1, respectively, from 3 animals each, p = 0.12) with a trend toward a ;10% reduction on LGI1 autoantibody treatment, similar to STED recordings in cultured neurons.In addition, we quantified Ca v 2.1 channel density at another LGI1-expressing synapse between dentate mossy fibers and CA3 neurons from chronically infused mice.Similarly, LGI1 autoantibodies did not affect Ca v 2.1 channel density at these synapses (eFigure 2).These data show that LGI1 autoantibodies caused, if anything, a small reduction in Ca v 2.1 channel density in boutons of hippocampal cultures and tissue.Therefore, alterations in calcium channels density cannot explain increased release probability by LGI1 autoantibodies.
LGI1 Autoantibodies Do Not Affect Presynaptic Calcium Channel Gating The LGI1-receptor ADAM22 has been shown to interact with various Ca v channel beta-subunits, 18 which in turn affect calcium current gating. 40We therefore determined the effect of LGI1 autoantibodies on calcium channel gating by directly measuring pharmacologically isolated presynaptic calcium currents in whole-cell voltage-clamp recordings from boutons in hippocampal cultures (Figure 3, A and B).Calcium currents on 3 ms depolarization were similar in amplitudes for control and LGI1 autoantibody-treated boutons (Figure 3C; median [IQR] current amplitude at 0 mV: 18.1 [10.5-28.4]pA and 18.3 [10.5-25.3]pA, n = 10 and 17, for control and LGI1, respectively, p = 0.66; 2-way ANOVA for overall effect: p = 0.079).In addition, the time course of current activation was not affected by LGI1 autoantibodies (eFigure 4, p = 0.86).Similarly, amplitude and time course of calcium current inactivation were unchanged following LGI1 autoantibody treatment (eFigure 4).Unaltered presynaptic calcium currents indicate that LGI1 autoantibodies did not affect calcium channel gating and thus cannot explain the increased release probability by LGI1 autoantibodies.

Nanoscale Localization of Presynaptic K v 1.1 and K v 1.2 Channels in Hippocampal Synapses
LGI1 interacts through ADAM22-receptors with K v 1 potassium channels, 17,18 which are reduced in animals treated with LGI1 autoantibodies. 27,30We therefore hypothesized that the release probability is increased because of the loss of presynaptic K v 1 channels.We first tested whether K v 1.1 and K v 1.2 channel subtypes, which have been linked to LGI1 functionally and in biochemical assays, 11,17,18 are localized presynaptically in cultured hippocampal neurons.Using SIM of neurons co-stained for Bassoon, we found that K v 1.1 and K v 1.2 channels were localized at presynaptic active zones in cultured hippocampal neurons (eFigure 5; Mander colocalization coefficients for K v 1.1 and Bassoon = 0.28 ± 0.11 (mean ± SD), n = 137 synapses; for K v 1.1 and Bassoon 0.62 ± 0.13, n = 195 synapses).To test potential limitations of the spatial resolution, we used postgelation expansion and immunolabeling in combination with SIM (Ex-SIM). 33amples expanded ;7.5-fold, thus enabling a spatial resolution of ;20 nm by multicolor SIM.Again, both K v 1.1 and K v 1.2 channels localized at vGlut1-positive presynaptic nerve terminals of cultured hippocampal neurons (Figure 4A and eFigure 5).Furthermore, both K v 1.1 and K v 1.2 were found inside and outside of the Bassoon-labeled active zone in cultured hippocampal neurons (eFigure 5).To corroborate the presence of K v 1 channels at hippocampal presynapses in brain tissue, we studied hippocampal dentate gyrus perforant path-granule cell synapses in perfusion-fixed hippocampal tissues.At these synapses, it was previously shown that LGI1 antibodies also increase the release probability. 27Using pre-embedding electron microscopy, we localized K v 1.1 channels (Figure 4B) and K v 1.2 channels (eFigure 5) at the perforant path-granule cell synapses.A three-dimensional reconstruction of perforant path axon terminals and the adjacent axons indicated a homogeneous K v 1.1 channel distribution (Figure 4C), consistent with <5% of both channel subtypes localized at or close to the small surface area building the presynaptic active zone (active zone and perisynaptic; Figure 4D and  eFigure 5).However, the large majority of the K v 1.1 and K v 1.2 channels were found outside of the active zone.Thus, complementary high-resolution light and electron microscopic techniques confirm the localization of both K v 1.1 and K v 1.2 channels at hippocampal presynapses and indicate a rather homogeneous distribution.
LGI1 Autoantibodies Reduce Presynaptic K v 1.1 and K v 1.2 Channels After we found presynaptic localization of K v 1.1 and K v 1.2 channels, we tested whether their localization was affected by LGI1 autoantibodies.
LGI1 autoantibodies were previously shown to reduce general synaptic K v 1.1 channels using confocal imaging 27 or western blots. 30We first investigated the co-localization of bound pathogenic LGI1 autoantibodies and K v channels using confocal and STED microscopy (eFigure 6).
LGI1 autoantibody localization showed a punctate pattern.Co-localization of LGI1 puncta with K v 1.1 and K v 1.2 at the soma and dendrites was weak, but the majority of LGI1 puncta had at least a weak K v signal, while many K v puncta had no LGI1 signal.Furthermore, we found that both K v 1.1 and K v 1.2 showed a strong signal at the axon initial segment (AIS), which was identified by the presence of Ankyrin G (AnkG) and a coincident lack of Microtubuleassociated protein 2 (MAP2; eFigure 7).The K v 1.1 and K v 1.2 staining in the AIS showed 190 nm spaced bands (eFigure 7) as described for other proteins at the AIS 41 ; however, LGI1 and K v colocalization at the AIS was weak.These results are not surprising because LGI1 might be secreted (but see ref. 42) and the interaction of LGI1 with K v 1 channels, possibly through ADAM-family proteins, is still not well understood. 14ur data thus argue against a fixed stoichiometric interaction of LGI1 with K v channels.
To analyze the effect of treatment with pathogenic LGI1 antibodies on K v 1.1 and K v 1.2 channels, we performed STED imaging because of its higher resolution compared with confocal microscopy and the higher throughput compared with the Ex-SIM technique.We investigated K v 1.1 and K v 1.2 channels at excitatory presynapses in cultured hippocampal neurons (Figure 5A).Because of the variability in K v 1 signal intensity between synapses, we repeated antibody application, immunostaining, image acquisition, and image analyses in 10 cultures independently generated from 10 different animals.
LGI1 autoantibodies reduced K v 1.1 (Figure 5B) and K v 1.2 signals (Figure 5C) at excitatory active zones by 10%-15% (median change for K v 1.1 within Bassoon −12.3%, K v 1.2 within Bassoon −10.2%, each p < 0.001 and n = ;5000 synapses).Similarly, K v 1.1 and K v 1.2 signals were reduced within the vGlut1-labeled presynaptic boundary (eFigure 8).Even when we analyzed each culture separately (which might represent an overcritical definition of the biological replicate), the LGI1 autoantibodies showed trends of reduction or statistically significant reduction of both, K v 1.1 and K v 1.2, within vGlut1 and Bassoon (eFigure 8).Consistent with a homogeneous distribution of K v 1.1 and K v 1.2 in electron microscopy, we also observed a reduction in the density of presynaptic K v 1.1 and K v 1.2 outside of the active zone (i.e., inside the vGlut1 but outside of the Bassoon mask;

particles (yellow spheres). (D)
Quantification of K v 1.1 particle localization within active zones (black), the perisynaptic space (dark gray; ≤60 nm from the active zone edge), and the extrasynaptic space (light gray; >60 nm from the active zone edge).Numbers in brackets indicate total particle counts or counts within respective localizations.
data not shown).Thus, presynaptic K v 1.1 and K v 1.2 channels are both homogeneously reduced within the presynaptic terminal on treatment with LGI1 autoantibodies.
LGI1 Autoantibodies Lead to Increased Presynaptic Action Potential Broadening K v 1 channels control presynaptic action potential duration (e.g., see ref. 42 and references therein).To determine the functional relevance of presynaptic K v 1 channel loss, we performed direct current-clamp recordings from boutons in hippocampal cultures 43,44 following autoantibody treatment.Action potentials evoked by current injections had large amplitudes and short half-durations (quantified as full-width recorded at half-maximal amplitude, FWHM), similar to previous findings at boutons of neocortical cultures. 43hanges in action potential shape were tested by evoking trains of 90 action potentials at 20 or 50 Hz (Figure 6A).LGI1 autoantibodies also increased bouton excitability, leading to aberrant action potential firing during current injections (eFigure 9; repetitive action potentials on prolonged current injections in 1/15 and 5/13 boutons for control and LGI1, respectively, p = 0.04).Similar to presynaptic action potentials, somatic action potentials were broadened and showed increased activity-induced broadening following treatment with LGI1 autoantibodies (eFigure 9).6][47] Thus, these data indicate that by reducing K v 1 channels, LGI1 autoantibodies enhance somatic and presynaptic action potential broadening and thus synaptic release probability.

Autoantibodies Targeting the EPTP Domain but Not the LRR Domain of LGI1 Cause Action Potential Broadening
To address which of the 2 main LGI1 domains is involved in the antibody-mediated action potential broadening, we again recorded somatic action potentials, this time however following treatment with patient-derived monoclonal autoantibodies specifically targeting only either the EPTP or the LRR domain (see supplementary material, eMethods, for details on the antibodies). 31We again first tested the colocalization of the pathogenic monoclonal LGI1 autoantibodies with K v 1.1 and K v 1.2 channels (eFigure 6).Similar to the polyclonal antibodies, there was little colocalization.In addition, there was little overall binding of the anti-EPTP antibodies.This is consistent with the original description of these antibodies in which the EPTP antibodies inhibited the docking of LGI1 to ADAM22/23 and induced pronounced memory defects, but the surface binding of EPTP antibodies was much lower compared with the LRR antibodies. 30Compared with cells treated with control isotype matching monoclonal antibodies, LRR autoantibodies did not affect action potential broadening during 20 Hz trains (Figure 7  trains (Figure 7, A-C and eFigure 10; median [IQR] FWHM of last 10 train action potentials at 20 Hz: 1.32 [1.12-1.46]ms for EPTP, n = 17, p = 0.027).The broadening of somatic action potentials following EPTP autoantibody treatment was similar in magnitude to the broadening induced by polyclonal LGI1 autoantibodies (cf. Figure 7, B and C and eFigures 9 and 10), suggesting that antibody binding to the EPTP domain underlies action potential broadening.

Discussion
Our results have important implication for understanding the pathophysiology of LGI1 autoimmune encephalitis and the physiologic functions of LGI1.In particular, our study demonstrates that (1) LGI1 autoantibodies broaden presynaptic action potentials, which explains the observed increase in release probability.(2) We did not find relevant changes in the density nor the gating of calcium channels on LGI1 autoantibody treatment.(3) The homogeneous presynaptic distribution and reduction of K v 1.1 and K v 1.2 channels on LGI1 autoantibody treatment indicate that LGI1 can act outside of the release site in addition to its transsynaptic function.( 4) Experiments with domain-specific patientderived monoclonal autoantibodies indicate that action potential broadening is mediated by autoantibodies targeting the EPTP domain but not by antibodies targeting the LRR domain.Thus, our study provides a mechanistic framework explaining the neuronal hyperactivity of patients with LGI1 antibody encephalitis.
It is controversial whether LGI1 autoantibodies affect synaptic transmission presynaptically, postsynaptically, or both, presynaptically and postsynaptically.We found that LGI1 autoantibodies decreased paired-pulse ratios and increased synaptic depression arguing for a presynaptic effect of LGI1 autoantibodies. 48Our results are consistent with previous  studies reporting that LGI1 autoantibodies increase synaptic strength and decrease paired-pulse ratio at hippocampal perforant path-granule cell synapses and reduce synaptic failures in CA1 neurons, 27 with a similar trend toward higher mEPSC frequency observed in CA3 neurons. 29Synapses onto hippocampal CA1 and CA3 neurons were not affected in strength or paired-pulse ratio by LGI1 autoantibodies, 25,27,28,30 which might be due to lower abundance of the LGI1 protein at these synapses. 17The increased release probability on LGI1 antagonism provides an explanation for the hyperactivity in both LGI1 autoantibody-treated neurons 25,28 and neurons of LGI1 knock-out mice. 11,20,21,49Furthermore, the increased release probability might also serve as a basis for the epileptic seizures of patients suffering from LGI1 antibody encephalitis. 50The faciobrachial dystonic seizures respond intriguingly fast to immunotherapy, whereas antiseizure medication is often ineffective. 4,51Our data suggest that the ineffectiveness of antiseizure medication could be due to the direct, antibodyinduced increase in presynaptic function.More studies are needed to better understand the underlying cause of seizures in anti-LGI1 encephalitis to develop effective causative and symptomatic treatment.
LGI1 and ADAM receptor proteins have previously been shown to affect K v 1 channel gating 17 and expression, 16,52 and LGI1 autoantibodies immunoprecipitate with K v channels. 3,15e found that both K v 1.1 and K v 1.2 subunits were localized presynaptically, consistent with previous results on K v 1 channel localization. 53LGI1 autoantibodies reduced presynaptic K v 1.1 and K v 1.2 channels, in agreement with reduced hippocampal K v 1.1 fluorescence 27 and K v 1 protein levels 30 following autoantibody treatment.Direct bouton patchclamp recordings revealed enhanced action potential broadening during train stimulation, a well-known consequence of reduced K v 1 conductance on activity-dependent K v 1 channel inactivation 45,46 or pharmacologic K v 1 channel block (e.g., see ref. 42 and references therein).Therefore, our presynaptic structural-functional analysis provides direct support for the following mechanistic steps: (1) LGI1 autoantibodies interfere with LGI1's endogenous function of increasing the presynaptic potassium channels density.(2) The reduction of presynaptic potassium channels prevents efficient repolarization of the presynaptic action potential.(3) The resulting longer presynaptic action potential increases release probability.
Consistent with increased release probability on antibody application, knock-out of LGI1 in mice increased transmission at hippocampal CA3-CA3 synapses 16 and in CA1 neurons. 21,54Furthermore, overexpression of LGI1 decreased synaptic strength at perforant-path granule cell synapses. 19he synaptic strengthening on LGI1 knock-out is probably mediated presynaptically by an increased release probability because LGI1 knock-out postsynaptically either decreased AMPAR clustering and quantal size 11,22,24,26 or did not affect quantal size. 19,21However, some synapses show no presynaptic effect on LGI1 knock-out or LGI1 application.For example, in CA1 neurons, PPR was mostly unaffected by LGI1 application 11 or LGI1 knock-out. 11,22,24,54These differences in the presynaptic effect of LGI1 knock-out on synaptic transmission may relate to the differential expression of LGI1, with highest expression in the hippocampal outer and middle molecular layers of the denate gyrus (perforant path-granule cell synapses). 17Furthermore, LGI1-overexpression shortened presynaptic action potentials in primary hippocampal cultures, leading to lower action potentialevoked calcium entry and hence glutamate release. 42Therefore, LGI1 autoantibodies induce effects that are reminiscent of those observed in LGI1 knock-out mice and thus support the mechanistic model that LGI1 increases the presynaptic potassium channel density, shortens the presynaptic action potential duration, lowers the release probability, and thereby dampens neuronal activity.
We found that K v 1 channels are homogeneously distributed across the axon and bouton and only a minority of potassium channels was located at the presynaptic release site (Figure 4 and eFigure 5).Furthermore, LGI1 autoantibodies decreased the K v 1 density within and outside of the Bassoon-labeled release sites, indicating a homogeneous reduction throughout the bouton (Figure 5 and eFigure 8).Our data therefore argue that LGI1, in addition to its transsynaptic alignment, controls potassium channels also outside of the release site.Indeed, it was recently shown that LGI1 autoantibodies also alter the K v 1 cluster distribution at the axon initial segment. 55,56The autoantibody-induced increase in neuronal excitability was mediated by antibodies specifically targeting the LRR domain. 29,30,55,56Consistently, structural analyses indicate that LGI1 can form protein complexes in a cis-configuration serving as an extracellular scaffold instead of a transsynaptic hub. 14,57It remains to be determined if the density of presynaptic K v 1 channel outside of the release site is controlled by LGI1 proteins in the cis-configuration.
The analyses of calcium channels were motivated by the increase in synaptic release probability following LGI1 autoantibody treatment, a phenomenon typically observed on changes in calcium channel density or function.Furthermore, proteome data previously indicated an interaction of the LGI1-receptor ADAM22 with pore-forming calcium channel alpha-subunits and their beta-subunits, 18,37 which control calcium channel surface expression and kinetics. 40,58We used STED and EM imaging of Ca v 2.1 calcium channels and direct electrophysiologic recordings of presynaptic calcium current density and gating kinetics.Yet, we found neither presynaptic Ca v 2.1 channel abundance nor calcium current amplitude and channel gating kinetics were strongly affected by LGI1 autoantibodies (if anything, there was a reduction in the channel density).Therefore, potential effects of LGI1 on presynaptic calcium channels do not contribute to the increased release probability induced by LGI1 autoantibodies.
Previously, patient-derived monoclonal autoantibodies were used to specifically target the EPTP or the LRR domain of LGI1. 30 Although EPTP-targeting autoantibodies led to enhanced broadening during train stimulation in our recordings, LRR autoantibodies did not affect action potential broadening.The EPTP domain of LGI1 has been shown to mediate binding to ADAM22, and EPTP-targeting autoantibodies hence prevented binding of LGI1 to ADAM22. 12,26,29,30oth EPTP and LRR autoantibodies reduced K v 1.1 protein levels in hippocampus-enriched solubilized brain lysates, but the effect seemed stronger with EPTP-compared with LRR autoantibodies. 30By contrast, some studies observed an increased neuronal excitability only with LRR but not with EPTP autoantibodies 55,56 or a stronger effect on excitability with LRR compared with EPTP antibodies. 29RR-targeting antibodies were previously shown to interfere with multimerization and cause internalization of the LGI1-ADAM22 complex. 12,29,30A differential effect of autoantibodies targeting EPTP and LRR is conceivable because of the complex interplay of various types of potassium channels in controlling excitability and action potential repolarization. 59However, more studies are needed to understand the differential effect of the subunit-specific autoantibodies on excitability and action potential repolarization.Furthermore, although we tested 2 monoclonal antibodies for each LGI1 functional domain, our data cannot rule out that LRR autoantibodies with different binding epitopes other than those tested here are able to affect presynaptic K v 1 function.Indeed, there might be differences with the subclones used in previous studies. 29,56owever, we use the exact same set of antibodies as in Sell et al., 55 who also found stronger effects with LLR antibodies on excitability as previous studies. 29,56It is difficult to rule out that due to technical reasons, the antibodies change their potency.However, our data (eFigure 6) argue against the possibility that the absence of action potential broadening with LRR antibodies is due to a lost binding ability of the antibodies.Taken together, the data thus suggest differences in the regulation of excitability and action potential duration by the 2 domains of LGI1, which could be reflected in differential symptoms associated with mutations in these domains, such as auditory features that occur less frequently in congenital epilepsy caused by EPTP truncation compared with LRR truncation. 60 , A and B; median [IQR] PPR at 20 Hz: 1.03 [0.84-1.24],0.85 [0.82-0.96],and 0.67 [0.53-0.69],n = 14, 19, and 11 for control, LGI1-1d, and LGI1-7d, respectively; nonparametric Kruskal-Wallis ANOVA test p < 0.001 and post hoc test p < 0.001 for control and LGI1-7d).

Figure 2
Figure2LGI1 Autoantibodies Have Little Effect on Presynaptic Ca v 2.1 Calcium Channel Density

Figure 3
Figure3LGI1 Autoantibodies Do Not Affect Presynaptic Calcium Channel Gating

Figure 4
Figure 4 Nanoscale Localization of Presynaptic Kv1.1 Channels in Hippocampal Synapses Action potential broadening was pronounced following LGI1 autoantibody treatment during 20 Hz train stimulation (Figure 6, B and C; 20 Hz: median [IQR] broadening of the last 10 action potentials: 29.1 [25.0-37.7]% and 48.4 [25.6-77.1]%, n = 17 and 15 for control and LGI1, respectively, p = 0.05) and 50 Hz train stimulation (p = 0.03; eFigure 9).Owing to the large bouton-to-bouton variability, the absolute duration of the last 10 action potentials only showed a trend toward an increase duration (p = 0.23 and p = 0.16 for 20 and 50 Hz, respectively; data not shown).In contrast to the duration of action potentials, the amplitudes of presynaptic action potentials were not affected by LGI1 autoantibodies (eFigure 9; change in median amplitude of last 10 action potentials <3% at 20 Hz and <10% at 50 Hz, both p > 0.05).Besides changes in action potential broadening, treatment with , A-C; median [IQR] FWHM of last 10 train action potentials at 20 Hz: 1.08 [0.98-1.20]ms and 1.20 [0.99-1.31]ms, n = 17 and 22 for control and LLR, respectively, p = 0.45) and 50 Hz trains (eFigure 10).By contrast, treatment with EPTP autoantibodies led to enhanced action potential broadening during 20 and 50 Hz

Figure 6 LGI1
Figure6LGI1 Autoantibodies Lead to Increased Presynaptic Action Potential Broadening

Figure 7
Figure 7 Autoantibodies Targeting the EPTP Domain but Not the LRR Domain of LGI1 Cause Action Potential Broadening